Induction of Noncatalytic TrkB Neurotrophin Receptors during Axonal ...

The Journal

Induction of Noncatalytic TrkB Neurotrophin Sprouting in the Adult Hippocampus Klaus D. Beck,’ Fabienne McNeill,’ Caleb E. Finch,’

Lamballe,* Riidiger Klein,* Mariano Barbacid,* Franz Hefti,’ and Jonathan R. Day3

of Neuroscience,

Receptors

September

1993,

13(g): 4001-4014

during Axonal

P. Elyse Schauwecker,’

Thomas

H.

‘Division of Neurogerontology, Andrus Gerontology Center, University of Southern California, Los Angeles, California 90089-0191, 2Department of Molecular Biology, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 085435842, and 3Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802-5301

Brain-derived neurotrophic factor (BDNF) and its signal transducing receptor, the TrkB tyrosine protein kinase, are expressed at high levels in the hippocampus of the adult brain, suggesting a role for BDNF mechanisms in neuronal plasticity. To test this hypothesis, we used defined lesions of perforant path and fimbria-fornix, two major hippocampal afferents, to remove synapses on dendrites of dentate gyrus granule cells and pyramidal cells of Ammon’s horn and induce synaptic rearrangements. These combined lesions remove afferent connections from entorhinal cortex and septum and produce massive sprouting of axons of the commissural/associational pathways into the molecular layer of the hippocampal dentate gyrus. At days 1, 3, and 6, the lesions decreased BDNF mRNA expression ipsilaterally to approximately 50% of control, with complete recovery at 14 d. The lesions did not alter t&B mRNA levels in neuronal layers of the hippocampus; however, they resulted in a pronounced induction of t&B mRNA expression in hippocampal non-neuronal cells 6-l 4 d after lesioning. The induction corresponded in time and place to the synaptic reorganization in the lesioned hippocampus. The mRNA species newly induced by the lesions corresponded to those transcripts encoding the noncatalytic TrkB receptor isoform that lacks the cytoplasmic protein kinase domain. Expression of mRNAs coding for neurotrophin-3 and the TrkC tyrosine protein kinase were not altered by the lesions. The findings suggest that truncated noncatalytic TrkB molecules expressed on the surface of glial cells play an important role in plasticity of the adult brain, possibly regulating the concentration of bioactive neurotrophins or the responsiveness of neurotrophin receptors. Alternatively, they may play a role in presenting neurotrophin molecules to growing axons. [Key words: hippocampus, astrocytes, angular bundle, fimbria, fornix, entorhinal cortex, septum, plasticity, brainderived neurotrophic factor, neurotrophin-3, trkB, trkC]

Received Dec. I I, 1992; revised Mar. 12, 1993; accepted Mar. 30, 1993. This study was supported by U.S. Public Health Service Grants NS22933, NS30426,AG10480,andAG07909,NSFGrantBNS-9021255,StateofCalifomia Alzheimer Research Program Contract 91-12965, and the National Parkinson Foundation. K.D.B. was supported by a postdoctoral fellowship from Fritz Thyssen Foundation, Germany, and a pilot grant from Alzheimer’s Association. Correspondence should be addressed to Klaus D. Beck, M.D., at the above address. Copyright 0 1993 Society for Neuroscience 0270-6474/93/l 3400 l-14$05.00/0

The ability of neurons to adapt or modify their structure in response to changes in intrinsic and/or extrinsic environmental cues, that is, neuroplasticity, is a fundamental prerequisite for the formation of the neuroanatomical networks during brain development, and is believed to remain important during adult function of specific brain regions, such as the hippocampus (Black et al., 1985; Purves and Lichtman, 1985). It has been suggested that many of the cognitive functions that are successfully maintained with aging depend on the ability of the hippocampus to preserve or repair its internal neural circuitry in response to naturally occurring cell loss and partial deafferentation. The lack of adequate neuroplasticity may underlie behavioral deficits as they occur in Alzheimer’s disease (Coleman and Flood, 1987; Cotman and Anderson, 1988). The adult hippocampus expresses high levels ofbrain-derived neurotrophic factor (BDNF) (Hofer et al., 1990; Phillips et al., 1990; Wetmore et al., 1990) and its receptor, the TrkB tyrosine protein kinase (TK) gp 1451~~~ (Klein et al., 1989, 1990a,b), which is likely to mediate the trophic effects of BDNF (Klein et al., 199 1; Soppet et al., 199 1; Squint0 et al., 199 1). In addition to gp14Y@, the mouse trkB locus encodes a noncatalytic receptor isoform, gp95”kb (Klein et al., 1990a). In this study, we hypothesize that the interactions between BDNF and its TrkB receptors may play an important role in modulating the plasticity of the adult hippocampus. BDNF, a member of the NGF family of neurotrophins, was purified as a survival-promoting factor for sensory neurons (Barde et al., 1982; Leibrock et al., 1989). Two novel members of this neurotrophin family, neurotrophin-3 (NT-3) (Ernfors et al., 1990a; Hohn et al., 1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al., 1990a; Rosenthal et al., 1990) and neurotrophin4/5 (NT-4/5) (Berkemeier et al., 1991; HallbiiGk et al., 1991; Ip et al., 1992), have been recently described. Among mammalian species, there is complete conservation of the 119 amino acid sequence of mature BDNF and approximately 50% identity with the other neurotrophins. BDNF synthesis occurs predominantly in cortical and hippocampal neurons (Hofer et al., 1990; Phillips et al., 1990; Wetmore et al., 1990). This is very different from NGF and the other neurotrophins that are expressed in many peripheral tissues. Also in contrast to the situation with NGF in the basal forebrain/hippocampal pathway, where the neurotrophic factor and its receptor molecules, ~75~~~~ and gp140’rk, are synthesized in target and responsive tissues, respectively (Thoenen et al., 1987, review; Merlio et al., 1992), BDNF-synthesizing hip-

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in the Hippocampus

pocampal and cortical cells also express mRNA coding for TrkB receptor protein (Klein et al., 1990b). These findings are compatible with the view that BDNF acts as an autocrine factor, rather than as a “classical” target-derived neurotrophic factor. BDNF mRNA levels are developmentally regulated in the rat brain (Maisonpierre et al., 1990b; Friedman et al., 199 1). In the adult brain, BDNF mRNA is elevated by seizures (Zafra et al., 1990, 199 1; Isackson et al., 199 1; Dugich-Djordjevic et al., 1992a,b), mechanical damage (Ballarin et al., 199 l), and ischemia (Lindvall et al., 1992), suggesting involvement of BDNF in mechanisms of neurodegeneration/protection. Experimental lesions of hippocampal afferents have been used to examine neuroplasticity in the hippocampus and the effect of aging on these processes (Cotman et al., 198 1; Cotman and Anderson, 1988; reviews). Unilateral ablation of the entorhinal cortex removes approximately 85% of the synapses in the outer two-thirds of the dentate molecular layer (Matthews et al., 1976a,b). The other major hippocampal target of entorhinal cortex afferents are the dendrites in the lacunosum-molecular layer of Ammon’s horn (Steward and Scoville, 1976). In response to entorhinal cortex lesion, synapses in the dentate molecular layer are replaced by new synaptic profiles from ;he surviving afferent fibers of the commissural/associational, septal, locus ceruleus, and crossed-perforant pathways. Predominantly, axon collaterals from the commissural/associational pathway expand outward and form new synaptic contacts (Lynch et al., 1976; Scheff et al., 1980), replacing the lost synapses of the most medial component of the perforant pathway (Lynch et al., 1976). New synaptic contacts from axons of septal, locus ceruleus, and crossed-perforant pathways are concentrated in the outer half of the dentate molecular layer (Lynch et al., 1972; Steward et al., 1974, 1976; Steward and Vinsant, 1983). Aged rats show delayed onset of this reactive synaptogenic response compared to young adults (Scheff et al., 1980; Hoff et al., 1982). Degeneration of the entorhinal neurons forming the perforant path represents one of several specific degenerative events in the hippocampus in Alzheimer’s disease. Besides the loss of perforant path axons, there is degeneration of cholinergic afferents from the septum and noradrenergic afferents from the locus ceruleus (Mann, 1985; Price, 1986; reviews). Geddes et al. (1985) and Gertz et al. (1987) reported that the pattern of anatomical reorganization in the Alzheimer hippocampus is qualitatively similar to that found in young adult rodent brains following a perforant path lesion, as indicated by changes in the intensity of AChE staining and the redistribution of kainic acid receptors in the dentate gyrus. However, the expansion of the commissural/association pathways into the outer two-thirds of the molecular layer of the dentate gyrus in Alzheimer’s disease, defined by the redistribution of kainic acid binding sites, is greater then that found in rats with unilateral perforant path transections (i.e., 57% vs 73% increase) (Geddes et al., 1985). These data suggest that, in Alzheimer’s disease, the effect of the loss of perforant path axons on plasticity of other axons may be enhanced by the concomitant depletion of inputs from the septum and locus ceruleus. Based on the hypothesis that combined lesions of hippocampal afferents more closely model the combination of neuroanatomical deficits of Alzheimer’s disease, such lesions were chosen in the present study for the analysis of BDNF mechanisms. In adult rats, lesions of the perforant path were combined with complete transections of the fimbria-fomix, which remove the majority of septal cholinergic axons and noradrenergic axons of the locus ceruleus (Walaas, 1983, review). We studied the effects

of such lesions on temporal and subregion-specific expression of BDNF and trkB-specific transcripts using quantitative in situ and Northern blotting techniques. These combined lesions result in precisely defined, complex changes of both BDNF and trkB mRNA levels, suggesting that BDNF mechanisms play a crucial role in synaptic rearrangements produced by hippocampal afferent lesions.

Materials

and Methods

and brain lesions. Male Fisher 344 rats (225-250 gm) were anesthetized with pentobarbital(75 mg/kg) and placed in a stereotactic apparatus (Kopf Inc., Tujunga, CA). Fimbria-fomix and perforant path transections, as described in detail elsewhere (Gibbs et al., 1985, 199 1; Anderson et al., 1986; Gomez-Pinilla et al., 1992), were done with an extendable Scouten knife (Kopf Inc., Tujunga, CA). Briefly, for fimbriafomix transections, the knife was lowered through a drill-hole in the skull to a position of 2.0 mm posterior and 2.0 mmlateral ofthe bregma and 5.0 mm below the level of the dura. The wire of the knife was then extended 2.0 mm toward the midline, and the cut was made by moving the extended blade up 4.0 mm. For perforant path (angular bundle) transections, a hole was drilled 5.3 mm lateral and 1.O mm rostra1 from lambda. The knife was lowered to 5.0 mm below dura, at an angle of 30” in posterior-medial direction (ear bars = 0”). The blade was extended 1.O mm and raised 4.0 mm. Wounds were sutured and the animals kept under standard housing conditions for various intervals of time. They were killed by decapitation, and brains were dissected and immediately frozen for further assays. In situ hybridization of BDNF, NT-3, t&B, and trkC mRNA. Brains of four rats for each time after lesion and control were frozen in isopentane at -25°C. Sections (14 pm) were cut on a cryostat, mounted on subbed slides, and stored at - 70°C. Immediately preceding the hybridization, slides were brought to room temperature and fixed for 30 min in 4% paraformaldehyde/lOO mM sodium phosphate buffer, pH 7.4, followed by three 10 min washes in PBS. Antisense 35S-labeled BDNF cRNAs were transcribed from a DGEM42 plasmid (Promega, Madison, WI) containing a 460 base pair (bp) rat BDNF insert (Phillips et al., 1990). A 35S-labeled riboprobe specific for NT-3 was transcribed from a 473 bp rat NT-3 fragment in a pGEM32 vector (Promeaa. Madison. WI). Three tvnes of Drobes derived from t&B cDNA cloneswere used to distinguish between transcripts encoding the catalytic gp145’+” and the noncatalytic gp951rkA receptors (Klein et al., 1989, 1990a): (1) a YS-labeled antisense trkB cRNA probe capable of detecting all known trkB transcripts (PAN) was transcribed from linearized pFRK16 (Klein et al., 1989) as previously described. pFRK16 is a pBluescript-derived plasmid containing a 483 bp insert of mouse tl.kB cDNA encoding a portion of the extracellular domain of gp145’rks and gp95”AB receptors (Klein et al., 1989); (2) a YS-labeled trkB TK+ cRNA probe specific for those trkB transcripts encoding the gp1451rkB tyrosine protein kinase receptor was transcribed from linearized pFRK29, a pBluescript-derived plasmid containing a 280 bp insert encoding the &OH-terminal region of the catalytic domain of gp145’,A”B(probe”D” in Klein et al.. 1990a): (3) a 35S-labeled trkB TK- cRNA nrobe snecific for those transcripts ‘&&ding the gp95‘rkRnoncatalytic receptors was transcribed from pFRK3 1, a pBluescript-derived plasmid containing a 250 bp insert derived from sequences specific to gp95”“8 transcripts (probe “E” in Klein et al., 1990a). A 33P-labeled cRNA probe specific for trkC gene transcripts was prepared from a pGEM3Z-derived plasmid containing a 570 bp DNA insert encoding a portion of the extracellular domain of the mouse TrkC receptors (Lamballe et al., 199 1). Synthesis of this trkC-specific cRNA probe was performed in the presence of 50 mCi of 33P-UTP (3000 Ci/ mmol; New England Nuclear) using the pGEM express core system kit (Promega, Madison, WI). Hybridization was performed at 50°C for 3.0 hr with 50% formamide, 4 x SSC (1 x SSC = 150 mM NaCl, 15 mM Na citrate), 5 x Denhardt’s, 1.O% SDS, 10% dextran sulfate, 250 pg/ml tRNA, 25 &ml polyA, 25 Kg/ml polyC, and 0.1 M dithiothreitol, and then with 20 pg/ml RNase at-37°C for 30 min. Hybridized slides were washed at 60°C for 1 hr in 50% formamide. 0.5 M NaCl. 50 mM sodium phosphate (pH 7.0) and 1% fl-mercaptoethanol. ’ To evaluate the magnitude ofthe changes in BDNF mRNA expression in the hippocampal CA3 region and the dentate gyrus, we used camputer-assisted videodensitometry of the hybridization signal on direct autoradiograms. Densitometric measurements of five areas within the Animals

The Journal of Neuroscience, September 1993, 13(9) 4903

Figure

I. Hippocampal BDNF mRNA expression after combined fimbria-fornitiperforant path transection: direct autoradiograms of in situ hybridization; complete brain sections and magnified hippocampal areas are shown. A and B, unlesioned control; C and D, 1 d after lesion; E and F, 3 d after lesion; G and H, 6 d after lesion; and I and J, 14 d after lesion. Lesioned side is marked with an arrow. At 1, 3, and 6 d the hybridization signal in CA3 on the side of the lesion is decreased. dg, dentate gyrus.

4004

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et al. - Induction

of Noncatalytic

BDNF

A fi$ B

mRNA

TrkB

in the

Hippocampus

IN CA3

lciO I 125

Receptors

T

The formation of reaction product was kept at a minimum not to interfere with the subsequent trkB TK- in situ hybridization, which was performed as described above. Northern blot assays of BDNF and trkB mRNA. Rats received unilateral transections of fimbria-fomix, angular bundle, and combined lesions (n = 6 per condition). Six days later they were killed together with six unlesioned controls of the same age. Brains were removed, and the hippocampi were dissected and immediately frozen on dry ice. Total RNA was extracted according to Chomczynski and Sacchi (1987). RNA was separated on duplicate denaturing 1% agarose gels (10 &lane) and transferred to nylon filters. 32P-1abe1ed trkB cRNAs from the TK+ and TK- clones were prepared and hybridized to the duplicate membranes overnight at 50°C in- 1.5 x SSPE (1 x SSPE = 150 rnM NaCl, 10 mM NaH,PO,. 1.0 mM EDTA. DH 7.4). 50% formamide. 1% SDS. 0.5% fat-frke Glk powder, 20 fig/ml sheared salmon sperm DNA, and 350 rg/ml yeast tRNA. The filters were washed to final criterion in 0.1 x SSC, 0.1% SDS at 75°C and exposed to x-ray film. Filters that were first hybridized with the TK+ trkB riboprobe were stripped and rehybridized with the TK- probe and vice versa. Integrated optical densities were collected by computerized videodensitometry (ImageMeasure, Microscience, Inc., Federal Way, WA). For those trkB mRNAs encoding the noncatalytic receptor,we chose the 7.5 kilobase (kb) band; for the full-length species, we quantified the 4.8 kb band recognized by the TK-- and TK+-specific probes, respectively. Optical densities of control and lesioned sides of all four groups were expressedas percentageof the mean level of unlesionedcontrols -+ SEM. Statistical significance was assessed with two-way analysis of

I

-

100

8 E !i

75 -

d i-i 2 z p z

so-

25-

OCONTROL

1 DAYS

B ziP

BDNF mRNA

3 AFTER

6

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IN DENTATE

GYMS

150I 125

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variance using the statistics softwareCRUNCH (Crunch SoftwareCorp., Oakland, CA).

% 8 k : Ii

I

ipsilat.

75 _ con1ralat.

d p2

50.

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25 .

CONTROL

1 DAYS

3 AFTER

6

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LESION

Figure 2. Quantification of BDNF mRNA in situ hybridization: videodensitometric analysis of the hybridization signal from direct autoradiographs. A, CA3; B, dentate gyrus. One brain section from each of four rats per time postlesion and control was used. For each section five areas in CA3 and dentate gyrus from both sides were measured. Results are expressed as means in percentage of the mean of left and right side of unlesioned control & SEM of four animals. Asterisks indicate statistically significant differences to contralateral side (p < 0.05, ANOVA).

dentate gyrus and the CA3 pyramidal layer ipsilateral and contralateral to the lesion were collected from each of four control and four combined lesioned rats at 1,3,6, and 14 d after surgery. The significance of changes was evaluated with two-way analysis of variance using the statistical software CRUNCH (Crunch Software Corp., Oakland, CA). Combined glial jibrillary acidic protein immunohistochemistry/trkB TK- mRNA in situ hybridization. Brain sections were prepared, stored, and fixed as described above. Diethyl pyrocarbonate-treated water was used for all solutions. For glial fibrillary acidic protein (GFAP) immunohistochemistry, sections were blocked for 30 min at room temperature with phosphate-buffered saline (PBS) containing 10% normal horse serum, 1% bovine serum albumin, 0.1 M dithiothreitol, 0.3% Triton X-100, and 100 U/ml recombinant RNase inhibitor (Promega, Madison, WI). After removing the blocking solution, sections were incubated for 90 min at room temperature with a monoclonal anti-GFAP antibody (Boehringer Mannheim, Indianapolis, IN), diluted 1:400 in PBS. After rinsing with PBS, bound antibody was visualized using a Vectastain biotin-avidin horseradish peroxidase kit (Vector, Burlingame, CA) with 1 mg/ml diaminobenzidine, 0.0 15% H,O, as substrates.

Results Combined lesions of fimbria-fornix and perforant path resulted in modest, transient changes of BDNF mRNA expression in hippocampal neurons. BDNF mRNA levels were assessed by densitometric analysis of direct autoradiographs from in situ hybridization. Within CA3 ipsilateral to the combined lesion, the expression of BDNF mRNA was reduced at days 1, 3, and 6 to, respectively, 46%, 49%, and 57% of control levels and returned to control levels at day 14 (Figs. 1, 2). No other significant changes were detected in BDNF mRNA expression in neurons of the ipsilateral and contralateral hippocampus. Perforant path and fimbria transection alone did not produce comparable changes in BDNF mRNA expression from day 1 to day I4 (data not shown). At all times studied, BDNF mRNA signals were confined to hippocampal neurons, as reported in unlesioned animals (Hofer et al., 1990; Phillips et al., 1990; Wetmore et al., 1990). There was considerable constitutive expression of trkB mRNA in unlesioned animals in the hippocampus and throughout the brain, confirming earlier studies (Klein et al., 1990b). A probe corresponding to a sequence encoding part of the extracellular domain of trkB and common to all known trkB mRNA species was used for our in situ hybridization studies (trkB PAN; see Materials and Methods). In the unlesioned hippocampus, trkB mRNA expression was strictly confined to dentate granule cells and pyramidal neurons of Ammon’s horn (Fig. 3). Combined fimbria-fomix/perforant path transections did not induce significant changes of trkB mRNA expression in any of the hippocampal neuronal populations. However, 6 and 14 d after lesion, in situ hybridization revealed high levels of trkB mRNA in the outer two-thirds of the dentate gyrus molecular layer and the lacunosum-moleculare layer of the hippocampus proper, ipsilateral to the lesion (Fig. 3). This newly induced trkB hybridization signal was not present in the contralateral hippocampus at all times studied after combined and single perforant path and fimbria transections (data not shown). Next, we studied the response of the trkB gene to fimbria-

The Journal

of Neuroscience,

September

1993.

13(9) 4095

Figure 3. Hippocampal trkB mRNA expression after combined fimbria-fornix/perforant path transection: direct autoradiograms of in situ hybridization with a trkB PAN probe that recognizes all known trkB transcripts; complete brain sections and magnified hippocampal areas are shown. A and B, unlesioned control; C and D, 1 d after lesion; E and F, 3 d after lesion; G and H, 6 d after lesion; and I and .I, 14 d after lesion. Lesioned side is marked with an arrow. There is widespread expression in the rat brain, especially in cortex and hippocampus. At day 6 (G, H) trkB is induced in molecular layer (sm) and lacunosum-molecular layer (s/m) on the lesioned side; this signal is weaker, but still present at day 14 (I, 4.

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Induction

A

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3oo,

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Figure 4. Northern blot hybridization analysis of trkB transcripts: hippocampal trkB expression in control (CO) tissue and in response to perforant path (ECL) and fimbria/fomix (FFL) transection alone and combined lesions (EUFFL) on day 6 after lesion. A, Comparison of lesion responses of trkB TK- (top) and trkB TK+ (bottom) mRNAs revealed a twofold increase in the abundance of the trkB TK- transcripts on the lesioned compared to control side of the hippocampus. trkB TK+ mRNA did not show any response to the three lesions. B, Hybridization pattern of the three trkB probes used in this study. trkB TK+ hybridizes to trkB transcripts encoding the tyrosine protein kinase gp1451rkB receptors; trkB TKdetects trkB mRNAs codingfor the noncatalyticgp9PB receptors;trkB PANrecognizes a portion ofthe mRNA shared by all known trkB transcripts. Six rats for each lesion and control were used. Data are expressed as means in percentage of the mean of left and right side of unlesioned control f SEM. Asterisk indicates statistically significant difference to contralateral side (p < 0.03, ANOVA).

fomix and perforant path transections separately and in combination on day 6 to determine if one of these afferent projections alone was responsible for the induction of trkB mRNA in the non-neuronal layers of the hippocampus. In control hippocampi, Northern blot analysis revealed various trkB mRNAs, corresponding to those described in previous studies suggesting the existence of transcripts encoding the various TrkB receptor isoforms (Klein et al., 1990a; Middlemas et al., 1991) (Fig. 4A,B). Two probes recognizing specifically mRNA species coding for the catalytic gp1451rkB(trkB TK+, 4.8 kb band) and noncatalytic gp9SrkB (trkB TK-, 7.5 kb band) receptors were employed to test whether the newly induced trkB mRNA in the molecular and lacunosum-molecular layers corresponded to receptors capable of mediating the trophic effects of BDNF (Fig. 4B). Densitometric quantification of these transcripts in total RNA from homogenates of entire hippocampi 6 d after combined lesions revealed a twofold increase in ipsilateral expression of trkB TK- mRNA (Fig. 4A). Smaller increases on the lesioned compared to control side were also observed after perforant path and fimbria-fomix transections alone; however, these changes were not statistically significant. These results suggest that the magnitude of the response to lesion was related to the extent of the deafferentation and not the disruption of a partic-

ular afferent projection. In contrast to the pronounced changes seen in the expression of trkB mRNAs encoding the noncatalytic gp9WB receptor, no change in trkB TK+ mRNAs encoding the signaling gp 145’rksreceptors could be detected in our Northern blot studies (Fig. 2A). In situ hybridization analysis of the expression patterns of mRNAs coding for the TrkB receptor isoforms confirmed the results obtained by Northern blotting techniques. Distinct signals were obtained in the molecular and lacunosum-molecular layers ipsilateral to the lesion with the trkB TK- but not the trkB TK+ probe (Fig. 5). As shown in Figure 6, dark-field analysis of emulsion autoradiographs revealed the cellular distribution of trkB TK- and TK+ mRNAs in hippocampi of rats 6 d after combined lesions. On the lesioned side, a high number of trkB TK- mRNA-expressing cells were present in the outer two-thirds of the dentate gyrus molecular layer and in the lacunosum-molecular layer of CA 1. In addition, a small number of labeled cells were scattered throughout the hippocampus. The level of expression in these cells, reflected by the density of silver grains, was relatively high when compared to the grain densities over neurons in CA1 and dentate gyrus. The expression over neuronal layers was identical on control and lesioned sides. However, and in contrast to the lesioned side, there were no


Figure 5. Hippocampal induction of t&B TK- mRNA in response to combined deafferentation. Direct autoradiographs of in situ hybridization with trkB TK--specific (A) and trkB TK+specific (B) riboprobes show that the lesion-induced signal in the hippocampus represents trkB TK- mRNA. Induced by the lesion, TK- trkB mRNA is expressed in the molecular (sm) and lacunosum-molecular (s/m) layers on the lesioned side (indicated by arrow). Adjacent brain sections 6 d after combined lesion.

labeled cells in the molecular or lacunosum-molecular layers of the control side. Analysis of silver grain densities over cell bodies revealed that cells in the molecular and lacunosum-moleculare layers of the lesioned side had grain densities approximately two times higher than cells in the neuronal layers (Table 1). No difference was seen between neuronal layer cells of lesioned and unlesioned sides in CA 1 and dentate gyrus. In contrast to the robust induction of mRNA coding for noncatalytic TrkB receptors, in situ hybridization with the trkB TK+-specific riboprobe revealed low silver grain densities throughout dentate gyrus and CA1 and there were no differences between lesioned and contralateral hippocampus. A combined method of GFAP immunohistochemistry and trkB TK- in situ hybridization revealed that hippocampal astrocytes were the source of the lesion-induced trkB mRNA. Figure 7 shows GFAP-positive astrocytes in the ipsilateral molecular layer 6 d after combined lesion with silver grain accumulations over their cell bodies. The hippocampal expression of NT-3 and trkC mRNAs is shown in Figures 8 and 9, respectively. Rat brains taken at 1, 3,6, and 14 d following combined transection of fimbria-fornix/ perforant path were sectioned and hybridized with riboprobes specific for mRNAs encoding NT-3 and the extracellular domain of the TrkC tyrosine kinase receptors. In confirmation of earlier studies, NT-3 was expressed in dentate gyrus, CA2, and the medial portion of CA1 (Ernfors et al., 1990a,b; Phillips et al., 1990). This pattern was unaffected by the lesion at the times studied, therefore, only sections from control and day 6 after lesion are shown (Fig. 8). In situ hybridization with the trkC riboprobe resulted in a strong signal over neuronal layers of the entire Ammon’s horn and dentate gyrus that was not altered at 1, 3, 6, and 14 d after combined hippocampal deafferentation.

Figure 9 shows autoradiographs from unlesioned control and 6 d after surgery. Sense riboprobes from all plasmids used in this study were produced and used for in situ hybridization. Analysis of emulsions of brain sections hybridized with these sense probes did not reveal any unspecific labeling (data not shown). Discussion Our findings indicate that combined fimbria-fornix/perforant path transections, which strongly reduce the number of afferents projecting into the dentate gyrus molecular layer and lacunosurn-molecular layer of Ammon’s horn, induce the expression of noncatalytic gp9YkB receptors on astrocytes in this deaffer-

Table 1. Quantification of TK - t&B mRNA in situ hybridization d after combined unilateral perforant path and fimbria-fornix transection

Day 6

Molecular and lacunosummolecular layers [grains/cell (% area)]

CA1 [grains/cell (% area)]

Dentate gyrus [grains/pm* (% area)1

Ipsilateral Contralateral

18.4 Z!Z0.3 -0

12.1 * 0.7 9.7 1- 2.3

5.7 k 0.1 4.7 * 1.0

6

Data are mean percentage area occupied by grains per cell f SEM for molecular and lacunosum-molecular layers, CAl, and dentate gyrus. Ten cells per side from each of four rats were analyzed. For the dentate gyms, data are expressedas mean percentage area k SEM (n = 4) occupied by grains in a 5067 k 14 1 and 4694 r?-230 pm2 area covering the entire granule cell layer of the ipsilateral and contralateral hippocampus, respectively. y No silver grain accumulations over cell bodies present.

4008


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TK +

TK-

Figure 6. Dark field of in situ hybridization of hippocampi at 6 d after combined lesion using trkB TK--specific (left) and trkB TK+-specific (right) antisense riboprobes. A and E, lesioned; C and G, unlesioned sides; B, F, D, and H, corresponding bright fields. A, The combined lesion induces ipsilateral trkB TK- expression in the outer two-thirds of the dentate gyrus molecular layer (sm) and the lacunosum-molecular layer (slm)

of Ammon’s horn. Also in stratum radiatum, between the cell bodies of CA1 pyramidal neurons and dentate gyrus granule neurons, a few scattered trkB TK- mRNA-expressing ceils can be seen. C, Expression on the unlesioned side is limited to the hippocampal neurons, which give only a weak hybridization signal. E and G, trkB TK+ transcripts are expressed in the CA1 pyramidal and dentate granule neurons. The signal is weak and not influenced by the lesion.

entated area. Time course and localization of the gp9SrkB-specific mRNA expression coincide with the massive axonal growth and increasing number of synaptic densities in the deafferented zones after entorhinal cortex lesion (Steward and Vinsant, 1983). Our findings suggest that the noncatalytic gp9SrkB receptors, expressed by astrocytes, play an important role in synaptic remodeling of the adult brain, possibly by regulating the concentration

of bioactive

neurotrophins

or the responsiveness

of sig-

nal-transducing neurotrophin receptors, or by presenting neurotrophin molecules to growing axons. The combined fimbria-fomix/perforant path transections utilized in this study interrupt two major afferent projections to the hippocampus. The response to perforant path transection alone involves expansion of the commissural/associational fiber system from the inner 30% of the molecular layer about 40 pm into the region previously occupied by the ipsilateral medial entorhinal cortex projections (Cotman et al., 198 1; Cotman and

Anderson, 1988; reviews). Concomitant expansion of the ipsilateral septal and contralateral entorhinal cortex afferents occurs in the most distal branches of the dendrites. In this study, the septal afferent component was eliminated by fimbria-fomix transection, leaving the commissural/associational and the contralateral perforant path afferents as only main afferents to restore synaptic contacts. Presumably, under these conditions, each surviving afferent system must establish new synapsesover a larger area. This may require more time to accomplish and/ or a greater reactive response by the participating cells as compared to the situation after single angular bundle or fimbriafomix transections. It seems surprising that the expression of BDNF, a growth factor expressed by the vast majority of hippocampal neurons, is not more strongly affected by the enhanced synaptogenic response produced by fimbria-fomix/perforant path transections, since available information indicates the BDNF mRNA in the


Figure 7. Localization of trkB TKmRNA in situ hybridization labeling to astrocytes stained with GFAP immunohistochemistry. Bright-field (A) and corresponding dark-field (B) of colabeling experiment show silver grain accumulations from trkB mRNA in situ hybridization over astrocyte cell bodies positive for GFAP in the ipsilateral molecular layer 6 d after combined lesion. Scale bar, 15 pm.

hippocampus of adult rats is regulated by various experimental manipulations. Hippocampal BDNF mRNA levels increase very strongly following seizures (Zafra et al., 1990, 199 1; Ernfors et al., 1991; Isacksonet al., 1991; Dugich-Djordjevicet al., 1992a,b), ischemia (Lindvall et al., 1992), and mechanical damage (Ballarin et al., 1991). Furthermore, hippocampal BDNF mRNA levels were elevated after injection of quisqualate into the septal area (Boatell et al., 1992; Lindefors et al., 1992). While these latter findings were interpreted as a result of activation of the septal input to the hippocampus, they also are compatible with the view that quisqualate produced seizure-like activation in the hippocampus. In all these studies, changes in BDNF mRNA expression in the adult hippocampus were found within the first

hours after treatment or injury; these times were much shorter than those chosen in the present experiments. In contrast to the increased BDNF mRNA expression reported after mechanical damage (Ballarin et al., 1991) in our studies BDNF mRNA levels were reduced in the CA3 region at 1, 3, and 6 d after lesion and remained constant in the dentate gyrus. While we are further investigating the time-dependent regulation of hippocampal BDNF mRNA expression by hippocampal afferents, the present study indicates that synaptic rearrangements in the dentate molecular layer can occur in absence of prolonged, concomitant changes in BDNF expression. Similar to BDNF, expression of trkB transcripts in hippocampal neuronal layers remained unchanged from day 1 to day

4010


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Figure 8. Hippocampal NT-3 mRNA expressionis not alteredby combined deafferentation:direct autoradiographsof NT-3 in situ hybridization

on brain sectionsfrom control (A, B) and 6 d following combined perforant path/fimbria-fomix transection(C, D). NT-3 expressionis limited to medial CAl, CA2, and dentate gyms (dn), a pattern that is not altered by the lesion at 1, 3, 6, and 14 d (only the section at 6 d is shown). The lesionedside is indicated by an &row.’ 14 after combined fimbria-fomix/perforant path lesions. Howcvcr, and representing the most significant finding of this study, we observed a robust induction of trkB mRNA in astrocjrtes within the outer two-thirds of the molecular layer and the lacunosum-molecular layer on the side ofthe lesion. These regions represent the primary dendritic target of the lesioned projections and experience the highest degree of deafferentation. The trkB induction reflects entirely the expression of mRNA coding for noncatalytic gp95’rksreceptors (Klein et al., 1990a). These TrkE receptors bind BDNF (Squint0 et al., 199 l), but their function must be different from that mediated by catalytic gp14SrkB receptors after stimulation by BDNF (Klein et al., 1991; Soppet et al., 1991; Squint0 et al., 1991). Previous studies have reported the presence of transcripts encoding NT-3 and TrkC in hippocampal neurons. NT-3 mRNA has been detected in pyramidal neurons of medial CAl, CA2, and dentate gyrus granular neurons of adult rats (Emfors et al., 1990b; Phillips et al., 1990). In adult mouse, trkC mRNA is expressed by hippocampal granular and pyramidal neurons (Lamballe et al., 1991). These findings suggest that NT-3 and TrkC could also be involved in trophic mechanisms regulating hippocampal plasticity. Our findings failed to confirm this hypothesis since in situ hybridization 1, 3, 6, and 14 d after combined deafferentation did not show any detectable change in the

level or localization of NT-3 and trkC mRNA expression in the hippocampus. The selective activation of gp14Ykks by BDNF (Klein et al., 1991; Soppet et al., 1991; Squint0 et al., 199 1) and the pronounced expression of BDNF mRNA in hippocampal cells (Hofer et al., 1990; Phillips et al., 1990; Wetmore et al., 1990) suggest that BDNF is the functionally important molecule binding to noncatalytic gp95’rkBreceptors. However, NT-3 and NT4/5 are also able to bind to gp145frkks(Berkemeier et al., 1991; Glasset al., 1991; Klein et al., 1991; Soppet et al., 1991; Squint0 et al., 199 1); thus, a potential role for these neurotrophins must also be considered. However, the absence ofany change in NT-3 and trkC mRNA levels in response to the combined lesion and the lack of detectable NT-4/5 mRNA expression in the hippocampus (K. D. Beck and F, Hefti, unpublished observation) make BDNF the most likely of the neurotrophins to be involved in hippocampal synaptic rearrangement. The colocalization of GFAP immunoreactivity and trkB TKin situ hybridization signal identifies reactive astrocytes in the hippocampus as the source of the newly induced transcripts encoding gp95’rkBreceptors. Reactive astrocytes are believed to play two roles in the compensatory synaptogenic response to hippocampal deafferentation. Within the first few days after lesion, lipid inclusions in the cytoplasm of astrocytes are con-


Figure 9. Hippocampal trkC mRNA expression does not change following combined deafferentation: direct autoradiographs of in situ hybridization with a riboprobe directed against a segment of trkC mRNA coding for part of the extracellular domain. A and B, control; C and D, 6 d after lesion. The signal is present over Ammon’s horn and dentate gyrus (dg); the expression is not affected at days 1, 3, 6, and 14 after the lesion (only the section at 6 d is shown). The lesioned side is indicated by an arrow.

sidered evidence of phagocytosis of degenerating neuronal constituents (Peters et al., 1991). The second role for reactive astrocytes is suggested from in vitro and in vivo studies indicating that astrocytes can promote axonal elongation and neuron survival and that astrocytes in culture are able to produce neuro-

trophins and can support axon growth (Bakhit et al., 1991; Lu et al., 199 1; Yoshida and Gage, 199 1, 1992). Our results suggest that reactive astrocytes may regulate axonal sprouting during reactive synaptogenesis by sequestering neurotrophins in regions undergoing reorganization. This is reminiscent of axotomy-induced expression

ofp75LNGFR by Schwann

cells in the PNS (Heu-

mann et al., 1987; Taniuchi et al., 1988). Similarly, ~75~~~~ expression by Schwann cells occurs in human peripheral neuropathies (Sobue et al., 1988). In animals with experimental peripheral nerve injury, p75LNGFRexpression is downregulated after regrowth of axons in the distal parts of the nerve and axonSchwann cell contact seems responsible for this regulation (Taniuchi et al., 1988). The p75LNGFRprotein does not contain a functional cytoplasmic domain, and it has been speculated that its appearance on Schwann cells during regeneration provides a substratum for NGF action on regenerating sensory and sympathetic axons (Sobue et al., 1988). This hypothesis is supported by the absence of p75LNGFR induction after transection of central cholinergic, NGF-responsive pathways that do not show sig-

nificant regeneration after axotomy (Montero and Hefti, 1988; Junard et al., 1990). Thus, the noncatalytic gp95’rk*Breceptors may serve similar functions in the hippocampus after afferent lesions and play an important role for the growth of axons of the surviving afferent projections. Hypothetical mechanisms by which noncatalytic gp9SrkBreceptors regulate axonal growth are illustrated in Figure 10. Astrocytic gp95’rks receptors could bind BDNF molecules and thereby regulate the concentration of BDNF available to sprouting responsive axons (I). The receptors either could be located on the membrane of astrocytes (Ia) or could be released in the extracellular space and bind BDNF (Ib). These receptors could reduce the concentration of bioavailable BDNF by binding excess BDNF or, alternatively, prolong the availability of BDNF by acting as a depot for this neurotrophin. Since neurotrophins occur as homodimers (McDonald et al., 1991), it can be speculated that these dimers contain two binding sites, one interacting with a receptor on glial cells and the other with a receptor on a growing axon (II). The gp95’rkBreceptors induced on astrocyte membranes could then serve as BDNF presenting molecules for responsive regrowing axons. The notion that gp9Pkks receptors play an important role in cell-cell recognition and adhesion has been suggested on the basis of comparative sequence analysis (Schneider and Schweiger, 199 1) and has been

4012


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Receptors

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: trkB TK-

II

IO. Hypothetical functions for gp9FB receptorsduring lesioninduced synaptic rearrangement.

Figure

demonstrated for DTrk, the Drosophila homolog of the mammalian Trk family of receptors (Pulido et al., 1992). The fimbria-fomix/perforant path transections used in this study represent one of the few known situations where significant axonal growth occurs in the CNS after injury. This axonal growth coincides in place and time with the induction of truncated trkB receptors in non-neuronal cells. Understanding of regulatory mechanisms of trkB expression may lead to novel approaches to facilitate regeneration/sprouting in other CNS areas. References Anderson KJ, ScheffSW, DeKosky ST (1986) Reactivesynaptogenesis in hippocampalareaCA 1ofagedand youngadult rats. J Comp Neurol 252~374-384. Bakhit C, Armanini M, Bennett GL, Wong WLT, Hansen SE, Taylor R (1991) Increase in glia-derived nerve growth factor following destruction of hiDDOCamDd neurons. Brain Res 560:76-83.

Ballarin M, EmforsP, Liniefors N, PerssonH (1991) Hippocampal damage and kainic acid injection induce a rapid increase-in mRNA for BDNF and NGF in the rat brain. EXD Neurol 114:35-43. Barde YA, Edgar D, Thoenen H (1982) I&lication of a new neurotrophic factor from mammalian brain. EMBO J 1:549-553. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A (1991) Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron 7:857-866. Black IB, Adler JE, Dreyfus CF, Jonakait GM, Katz DM, LaGamma EF. Markev KM (1985) In: Neuroscience (Abelson PH. Butz LE. Snyder SH: eds), pi 30-j9. Washington, DC: American .&sociation for Advances in Science, publication no. 84- 13.

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growth factor synthesis and secretion by astrocytes. Brain Res 538: 118-126. Yoshida K, Gage FH (1992) Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines. Brain Res 569: 14-25. Zafra F, Hengerer B, Leibrock J, Thoenen H, Lindholm D (1990) Activity dependent regulation of BDNF and NGF mRNAs in the rat

hippocampus is mediated by non-NMDA glutamate receptors. EMBO J 9:3545-3550. Zafra F, Castren E, Thoenen H, Lindholm D (199 1) Interplay between glutamate and gamma-aminobutyric acid transmitter systems in the physiological regulation of brain-derived neurotrophic factor and nerve growth factor synthesis in hippocampal neurons. Proc Nat1 Acad Sci USA 88:10037-10041.